Acid hydrolysis of proteins



Dec. 18, 1951 F. L. RIGBY ACID HYDROLYSIS 0F PROTEINS Filed Aug. 28, 1948 ACID SUPPLY SAMPLING TUBE Inventor FRA/vc/s [Low R/aaY A as/v-r Patented Dec. 18, 1951 UNITED STATES PATENT OQFFICE .4011) HYDROLYSIS or rao'rnms Francis L. Rigby, Toronto, Ontario, Canada Application August 28, 1948, Serial No. 46,819

6 Claims.

This invention relates to an improved process for the acid hydrolysis of proteins while preventing tryptophane destruction.

At the present time there are three known, general methods of hydrolyzing proteins to obtain amino acids. Enzymatic hydrolysis is usually slow and incomplete, alkali hydrolysis racemizes the optically active amino acids, and acid hydrolysis is accompanied by tryptophane destruction and formation of a black or brownishblack substance, termed humin. Despite its disadvantages, the most commonly used of these methods is acid hydrolysis, chiefly because Of its rapidity and convenience. However, hydrolysates for oral administration are sometimes prepared by enzymatic hydrolysis since this obviates the necessity of replacing the tryptophane destroyed by acid hydrolysis.

In hydrolyzing proteins to produce amino acids, it is obviously desirable to obtain the maximum yields as rapidly as possible and to hydrolyze with the minimum amount of humin formation. The most rapid and satisfactory method yet found for hydrolyzing proteins is by the use of a strong acid, i. e., sulfuric or hydrochloric acid, even though such strong acid hydrolysis usually produces considerable humin and destroys a large amount of tryptophane, making it necessary for the manufacturer to add to his product tryptophane from an outside source, which procedure is both costly and inconvenient.

An object of this invention is to provide a novel method for the acid hydrolysis of proteins whereby humin formation and degradation of the naturally occurring tryptophane are minimized.

Another object of my invention is to establish a novel method of manufacturing tryptophane. Further objects of my invention will become apparent hereinafter.

My invention essentially comprises hydrolyzing a proteinaceous material with a hot mineral acid while simultaneously electrolytically reducing the reaction mixture by passing an electric current through it. I have found that this method gives a better quality of protein hydrolysate with a minimization of the humin formed, and without undue destruction of the tryptophane content. The resulting protein hydrolysate is free from insoluble humin, practically free from soluble humin, and contains a large proportion of the typtophane content of the original protein. Thus this hydrolysate requires little or no fortification with additional tryptophane, and indeed this hydrolysate is suitable as a source of tryptophane, from which the latter can readily be &0

2 lated. Furthermore, the hydrolysate is almost odorless and colorless and is much superior in flavor to the malodorous and unpleasant-tasting enzymatic hydrolysates.

The application of electrolytic reduction to the acid-protein mixture during the hydrolysis can be conveniently eflected by placing the mixture in the cathode compartment of an electrolytic cell wherein the anode is separated from the cathode by a diaphragm. Operation of the electrolytic cell results in the production of nascent hydrogen at the cathode and thus when. the acidprotein mixture is heated and stirred, the hydrolysis proceeds in a reducing atmosphere. Preferably the reaction mixture is brought to boiling slowly so that all of the hydrogen can sweep all of the oxygen from the cathode compartment before rapid hydrolysis begins. After completion of the hydrolysis, the reaction mixture is removed from the cathode compartment of the cell. This protein acid hydrolysate is then converted to a nutritionally valuable preparation in conventional manner by removal of the anions of the hydrolyzing acid, for example as described by Sahyun, Outline of the Amino Acide and Proteins, 1944, pp. 88-89.

The electrolytic cell for the hydrolysis should be one having the following features: (a) provision for heating and stirring or agitating the mixture; (b) reflux condenser to return volatil ized liquid to the cell; and. (0) separate outlets above the anode and cathode compartments for conducting away the gases produced by the electrolysis.

Referring now to the drawing, there is shown schematically an apparatus in which the process herein disclosed may be successfully practiced. A reaction vessel l of suitable construction is provided with, and communicates with, three upstanding tubes 3, 4, and 5 and around these tubes there are placed suitable condensers 6, I, and 8, respectively. The condensers are supplied with appropriate connections for inlet and outlet of cooling liquid. Any convenient agitator is provided within the reaction vessel, one available form comprising a shaft Ill through the central upstanding tube 4, supported by conventional bearings of which the lower one at If is capable of freely passing gases generated within the reaction vessel, and provided with a suitable stufllng box II at the upper end. The agitator is driven in any suitable manner, such as by a motor at the upper end of the shaft l0, and has conventional blades 1 3 at its lower end.

The lower end of the upstanding tube 3 communicates with a chamber defined by a porous diaphragm I5 which may be of any suitable material such as aluminum oxide. Within said chamber is a platinium anode IS. A cathode ll of large area, such as a lead wool mat, covers a substantial portion of the bottom of the reaction vessel directly below the tube 5. Electrical connectors and 2| connect the electrodes through an ammeter 22 and a variable resistance 23 to a battery 24, or other low voltage, direct current, power source.

An acid reservoir 26 is connected through a suitable valve to the upstanding tube 5 at a point below the condenser 8. A sampling tube 21 is provided to withdraw, as by siphoning, samples from within the reaction vessel in any conventional manner. A suitably heated tank 3|] is provided to contain anoil bath for heating the reaction vessel and its contents.

The details of the cells are not critical to my invention and the apparatus may be altered in numerous ways. For example, if the hydrolysis is carried out under pressure at temperatures above the boiling pointat atmospheric pressure, with simultaneous electrolytic reduction, less concentrated acids can be employed and the time required for the attainment of complete hydrolysis of the protein is greatly decreased. If a slight pressure of one to two pounds per square inch is desired, the apparatus can be suitably modified by imposing pressure from a head of water. If

a higher pressure is desired, for example, a pressure of about thirty pounds, a lead-lined metalreaction vessel can be used, in which case the lead lining can serve as the cathode of the electrolytic ce 1.

Any of the non-oxidizing acids commonly employed in the hydrolysis of proteins may be used in my process. The most useful reagents for this purpose are hydrochloric acid and sulfuric acid, but other non-oxidizing acids of sufliciently strong acidity are also satisfactory. Sulfuric acid is preferably employed due to its high rate and degree of hydrolysis of the starting protein and since it leads to formation of an innocuous gas, 1. e., oxygen, in the anode compartment of the electrolytic cell. When a lead cathode is used. sulfuric acid has several further important advantages in that (a) lead sulfate, which is formed at the cathode during the operation of the cell, has a very low solubility in sulfuric acid: (b) the removal of sulfate ions from the hydrolysis product can be effected easily by well-known procedures; and, (c) isolation of tryptophane from the hydrolysate, if'desired. is facilitated since in commonly practiced procedures the tryptophane is isolated from a solut on of sulfuric acid. The preservation of tryptophane is apparently favored by a preliminary hydrolysis period of at least about one hour using dilute acid of a normality up to about 1, followed by addition of more concentrated acid of normality of at least about 6 for the latter stages of the hydrolysis, the entire hydrolysis being carried out with simultaneous electrolysis. Hydrochloric acid may be used safely if the anode and cathode compartments are separated and provision made for the separate conduction of the hydrogen and chlorine out of the system. If such procedure requiring a diaphragm is to be used. the diaphragm is preferably constructed of aluminum oxide, unglazed porcelain or similar acid-resistant porous material.

Because of the highly acid nature of the hydrolyzing medium, the choice ofelectrode materials for use in the cell is confined to carbon and to 4 acid-resistant metals low in the electromotive series. In general, it will be found that lead, lead-mercury amalgam, platinum, and graphite are the materials of choice for the cathode, and that platinum is the most suitable for the anode. Generally speaking, increasing the cathode area and the current density results in increased tryptophane retention and smaller amounts of humin in the protein hydrolysate. However, a maximum level of tryptophane retention and humin prevention is soon attained, whereafter further increases in current density or cathode size produce negligible improvement. The actual values for this limit will of course vary somewhat according to the nature of the cathode material and also according to the construction of the electrolysis apparatus. If the current density is quite high during the early stages of the hydrolysis, the.

large volume of hydrogen produced may cause foaming of the reaction mixture, and for this reason addition of a small amount of an antifoam agent to the reaction mixture may be advantageous. For apparatus of laboratory size, the optimum size of a lead wool cathode is approximately 200 grams (calculated area of about 500 square centimeters) and the optimum aproximate current density is 0.8 ampere per square decimeter of cathode surface. The size of the anode is of little importance, provided that it is not so small that it limits the current flow below the optimum level.

Tryptophane recovery and humin prevention are inversely related to the proteinzacid ratio. The destruction of tryptophane and the reduction of the agent or agents responsible for its destruction are two competing reactions. Increasing the amount of protein increases the rate of tryptophane destruction to a greater extent that it increases the rate of reduction of the destructive agent since the concentration of the reducing agent, nascent hydrogen, remains unchanged. For example, the data set forth in the following table show the variation in tryptophane recovery obtained when 25 g. to 200 g. of casein are hydrolyzed, with simultaneous electrolytic reduction, by 1500 cc. of 6 N sulfuric acid, the volume of acid per g. of casein being varied from 60 cc. to 7.5 cc., or as it is commonly expressed, the proteintacid ratio ranges from 1:60 to 127.5.

TABnn I Effect of proteinzacid ratio Tryptophane, Per Cent 01 Original Hydrolysis Time, Hours Ratio, Ratio, Ratio, Ratio,

0.5 90.1 86 5 84. 9 82. 0 1.5 81.8 74.8 2. 0 68. 0 2. 5 i 70. 1 4. 0. 68. 0 62. 5 61. 0 57. 7 5. 5. 63. 4 59. 0 56.1 50. l 8.0. 61. 0 56. 4 52. 8 48. 0 l0. 0 59.1 55. 6 51. 7 46.1

The following examples are given to illustrate the practice of my invention, but are not to be construed as limiting.

EXAMPLE 1 tilized liquid to the cell; and (d) outlets for removing the gases produced by the electrolysis.

The mixture is boiled for fourteen hours, during which time continuous electrolysis is provided by a curr'entof 4 amps. at an electromotive force of 12 volts, as supplied by two six-volt batteries.

The. hydrolysis mixture becomes lightpink in color as the boiling point is approached. The pink color darkens to a light brown during the first hour of boiling and then disappears except for colloidal particles of fat; the hydrolysate is thereafter quite clear and transparent and has a light yellow or very lightamber color. The pervcentage of the original tryptophane. as measured.

by the colorimetric glyoxylic acid method of Shaw and McFarlane [Can. J. Research, B,'16.

361-368 (1938) l, and the degree of hydrolysis can be determined at intervals, ifdesired,'by with drawing samples from the reaction mixture. The

course of the hydrolysis as found by such tests is set forth in the following table: a

' TAsLI II Hydrolysis with simultaneous electrolysis Tryptophane, Hydrolysis,

Hydrolysis Time, Hours P3522150! Permcotglt of 0. 5 95. 14.1 1. 83. 0 57. 2 2. 5 71. 0 75.1 4. 0 86. 0 86. 0 6. 6 59.4 91.4 8. 0 66. 6 06. 8 i0. 0 54. 0 W. 7 l2. 0 53. 0 g. 2 l4. 0 52. 4 9

B. In contrast with the above results. when 100 g. of casein is hydrolyzed with 1500 ml. of 6 N sulfuric acid under the same heating conditions as in the above experiment but without electrolyzing conditions, the reaction proceeds as set forth in the following table:

TABLE III Control experiment Tryptophane, Hydrolysis, Hydrolysis P er cent of Per cent of original mm 1. 0 64 25. 7 1. s 51 sea 2. 5 48 74. 6 4. 0 40 86. 1 5. 5 28 92. 2 7. 6 21 96. 2 l0. 0 U. 1 13.0 0 no. 2

In addition to the extensive destruction of 'tryptophane as shown by the data in Table III.

the hydrolysate contains a considerable amount of insoluble humin and the filtered hydrolysateis black in color.

EXAMPLE 2 A. A mixture of 100 grams of commercial casein (obtained by lactic acid precipitation; nitrogen content, 13.3 percent, tryptophane content, 1.06 percent) and 1020 ml. of 0.5 N sulfuric acid is placed in the electrolysis apparatus mentioned in Example 1, and electrolysis is provided continuously during the following hydrolysis procedure. The mixture is heated to boiling and flve hours after boiling begins, the dropwise addition of 485 ml. oi 18 N sulfuric acid to the boiling reaction mixture is begun. This strong acid is reduced pressure to about ml.

' 0 added over aperiod of flvelioura. After a total of sixteen. hours, the hydrolysis is discontinued. The coursefof the hydrolysis is shown in the following table, these" data being obtained by analy- B. In the above experiment, when the pre-' liminary hydrolysis is shortened to one hour. the tryptophane recovery. is diminished only slightly, as showni'n the following table:

TASLI V Preliminary reduction for one hour in 0.5 N acid Tryp'to 11m, ew" s i are 00 5.25 81 no u 045. 71 11.50 m

C. Isolation of tryptophane.-'I'hree hundred and fifty grams of casein is hydrolyzed in a manner similar to that described in part B, using a two-hour preliminary hydrolysisin dilute sulfuric acid and a-subsequent- 12 hour hydrolysis in 6 N sulfuric acid, with simultaneous electrolysis of the reaction mixture during the entire hydrolysis. After completion of the hydrolysis period, the hot acid hydrolysate is siphoned directly into a sufficient volume of ice and water to give a final solution of 7% sulfuric acid. To this solution is added a solution of g. of mercuric sulfate in 7% sulfuric acid. This reaction mixture is placed in a refrigerator for 24 hours. The supernatant liquid is siphoned from the precipitated solid, which is then collected on a filter and washed with 7% sulfuric acid and with water. ,The washed solid is sus: pended in distilled water and barium hydroxide added until the solution is permanently alkaline to phenolphthalein. The suspension is .me-

1 chanically stirred and a stream of hydrogen sulflde is passed in until the mercury is completely precipitated. The mercury sulfide is filtered of! and sulfuric acid is added to the filtrate insufllcient amount to precipitate the barium as barium sulfate. The barium sulfate is illtered of! and the filtrate is concentrated under This concentrate is extracted with n-butyl alcohol in a continuous liquid-liquid extractor under reduced pressure for thirty-six hours. The water is removed from the butyl alcohol extract by repeated vacuum distillations followed by additions of pure butyl alcohol, until no further 7 precipitate is obtained. The crude product which contains tryptophane is crystallized from 60% ethyl alcohol. There is thus obtained 1.73 g. of crystalline l-tryptophane, representing 0.49% of the weight of casein used.

EXAIWPLE 3 One kilogram of commercial casein (obtained by lactic acid precipitation; nitrogen content, 13.3%; tryptophane content, 1.06%) is subjected to the purification procedure of Van Slyke and Baker [New York Ag. Exp. Sta., Geneva Tech. Bull. No. 65 (1918)]. This purification consists of two reprecipitations, thorough washing with water, one washing with ethanol, and a final washing with ether. The product contains 14.5% nitrogen and 1.20% tryptophane.

when 100 g. of the purified casein thus obtained is substituted for the 100 g. of commercial casein in the hydrolysis process described in Example 2, the following results are obtained, as set forth in Table VI:

TABLE VI Hydrolysis of purified casein 'Iryptophane g g g Per cent of 1 Original EXAMPLE 4 One hundred g. of purified casein, obtained as described in Example 3, is hydrolyzed with 1020 ml. of 0.5 N sulfuric acid for two hours. with simultaneous electrolysis, and the hydrolysis is then continued in 6 N sulfuric acid, also with simultaneous electrolysis, in a manner similar to that described in Example 2. The course of the hydrolysis is set forth in the following table:

TABLE VII Hydrolysis of purified casein with preliminary electrolysis in 0.5 N acid Tryptopliane Hydrolysis Per cent of Time, Home Original EXAMPLE Tun: VIII Hydrolysis of blood fibrin 'Iryptophane Hydrolysis Hydro] is Per Cent 01'' Per Cent oi Original Total EXAMPLE 6 One hundred grams of commercial wheat gluten (nitrogen content, 13.3 percent) is hydrolyzed in 1500 ml. of 6 N sulfuric acid with simultaneous-hydrolysis. The hydrolysate thus obtained contains no insoluble humin. In contrast, when this protein is hydrolyzed in a similar manner in sulfuric acid, without simultaneous electrolysis, insoluble humin amounting to 2.4 percent of the weight of wheat gluten used is formed.

EXAMPLE '7 One hundred grams of skim milk powder is hydrolyzed in 1500 ml. of 6 N sulfuric acid with simultaneous electrolysis. The hydrolysate thus obtained contains only traces of insoluble humin, whereas when skim milk protein is hydrolyzed in a similar manner without simultaneous electrolysis, the formation of insoluble humin exceeds 5 percent of the weight of protein employed.

It will be appreciated that'my process is applicable to any type .of proteinaceous material, whether of animal or plant origin, e. g., lactalbumin, casein, blood fibrin, wheat gluten, egg albumin, soy bean protein, et cetera.

I claim:

1. In a process for the acid hydrolysis of protein material to produce a mixture of amino acids containing a high proportion of the naturally occurring tryptophane originally present in the protein material, with minimization of humin formation, the step comprising hydrolyzlng the protein material by heating it in the cathodic compartment of a diaphragmed electrolytic cell with an aqueous solution of an acid, said acidic solution being non-oxidizing with respect to said protein material and hydrolysis products thereof and being of sufiicient concentration and said heating being of sufficient duration to largely hydrolyze the protein material, and simultaneously electrolytically reducing the hydrolysis mixture in said cathodic compartment by passing an electric current therethrough.

2. In a process for the acid hydrolysis of protein material to produce a mixture of amino acids containing a high proportion of the naturally occurring tryptophane originally present in the protein material, with minimization of humin formation, the step comprising hydrolyzing the protein material by heating it in the cathodic compartment of a diaphragmed electrolytic cell with an aqueous solution of an acid of a normality up to about 1 for a period of at least one hour, then adding acid of a normality of at least 6, and continuing the heating during a period of sufficient duration to largely hydrolyze the protein material, said acidic solution being nonoxidizing with respect to said protein material and hydrolysis products thereof, and simultaneously electrolytically reducing the hydrolysis mixture in said cathodic compartment by passing an electric current therethrough.

3. A process for the hydrolysis of protein material to produce tryptophane which comprises: hydrolyzing the protein material by heating it in the cathodic compartment of a diaphragmed electrolytic cell with an aqueous solution of an acid, said acidic solution being non-oxidizing with respect t said protein material and hydrolysis products thereof and being of suflicient concentration and said heating being of suflicient duration to largely hydrolyze the protein material, and simultaneously electrolytically reducing the hydrolysis mixture in said cathodic compartment by passing an electric current therethrough; neutralizing the hydrolysis product; and separating the tryptophane thus produced.

4. The process of claim 1, wherein the simultaneous hydrolysis and reduction is carried out above atmospheric pressure.

5. The process of claim 1, wherein the hydrolysis is conducted at about reflux temperature.

6. The process of claim 1, wherein the acid employed i's sulfuric.

FRANCIS L. RIGBY.

REFERENCES crrED' The following references are of record in the file of this patent:

UNITED STATES PATENTS OTHER REFERENCES Koperina et al.: J. Gen. Chem. (U. S; S. B), vol. 17 (1947), pp. 1651-55.

Lewis et a1.: J. Phys. Chem, vol. 43 (1939), 

1. IN A PROCESS FOR THE ACID HYDROLYSIS OF PROTEIN MATERIAL TO PRODUCE A MIXTURE OF AMINO ACIDS CONTAINING A HIGH PROPORTION OF THE NATURALLY OCCURRING TRYPTOPHANE ORIGINALLY PRESENT IN THE PROTEIN MATERIAL, WITH MINIMIZATION OF HUMIN FORMATION, THE STEP COMPRISING HYDROLYZING THE PROTEIN MATERIAL BY HEATING IT IN THE CATHODIC COMPARTMENT OF A DIAPHRAGMED ELECTROLYTIC CELL WITH AN AQUEOUS SOLUTION OF AN ACID, SAID ACIDIC SOLUTION BEING NON-OXIDIZING WITH RESPECT TO SAID PROTEIN MATERIAL AND HYDROLYSIS PRODUCTS THEREOF AND BEING OF SUFFICIENT CONCENTRATION AND SAID HEATING BEING OF SUFFICIENT DURATION TO LARGELY HYDROLYZE THE PROTEIN MATERIAL, AND SIMULTANEOUSLY ELECTROLYTICALLY REDUCING THE HYDROLYSIS MIXTURE IN SAID CATHODIC COMPARTMENT BY PASSING AN ELECTRIC CURRENT THERETHROUGH. 